Bottom Line:
Induction of gene expression in yeast and human cells involves changes in the histone modifications associated with promoters.A truncated H3 product is not generated in yeast carrying a mutation of the endopeptidase recognition site (H3 Q19A L20A) and gene induction is defective in these cells.These findings identify clipping of H3 tails as a previously uncharacterized modification of promoter-bound nucleosomes, which may result in the localized clearing of repressive signals during the induction of gene expression.

ABSTRACTInduction of gene expression in yeast and human cells involves changes in the histone modifications associated with promoters. Here we identify a histone H3 endopeptidase activity in Saccharomyces cerevisiae that may regulate these events. The endopeptidase cleaves H3 after Ala21, generating a histone that lacks the first 21 residues and shows a preference for H3 tails carrying repressive modifications. In vivo, the H3 N terminus is clipped, specifically within the promoters of genes following the induction of transcription. H3 clipping precedes the process of histone eviction seen when genes become fully active. A truncated H3 product is not generated in yeast carrying a mutation of the endopeptidase recognition site (H3 Q19A L20A) and gene induction is defective in these cells. These findings identify clipping of H3 tails as a previously uncharacterized modification of promoter-bound nucleosomes, which may result in the localized clearing of repressive signals during the induction of gene expression.

Figure 3: The H3 endopeptidase is a serine protease.(A) Inhibition of the H3 endopeptidase activity.The endopeptidase activity on recombinant H3 was tested in the presence of different proteases inhibitors. The reactions were analysed by Western Blot with anti C-terminal H3 antibody.(B) Protease inhibitor profile of the H3 endopeptidase activity.

Mentions:
As an alternative approach to determine the identity of the enzyme, we performed the cleavage reactions in the presence of inhibitors specific for different families of proteases (Figure 3). This screen shows that the serine protease inhibitors PMSF and leupeptin inhibit the activity to different extends (Figure 3A, lanes 5 and 9). Given that some cysteine proteases can also be inhibited by these compounds, we tested more cysteine protease inhibitors but none of them blocked the activity (Figure 3B). Furthermore, Figure 3A shows that pefacbloc SC plus, which reacts only with the nucleophilic serine in the catalytic site of proteases, can robustly inhibit the H3 endopeptidase activity (lane 12). Together, these results strongly suggest that the activity purified from yeast extracts is a serine protease.

Figure 3: The H3 endopeptidase is a serine protease.(A) Inhibition of the H3 endopeptidase activity.The endopeptidase activity on recombinant H3 was tested in the presence of different proteases inhibitors. The reactions were analysed by Western Blot with anti C-terminal H3 antibody.(B) Protease inhibitor profile of the H3 endopeptidase activity.

Mentions:
As an alternative approach to determine the identity of the enzyme, we performed the cleavage reactions in the presence of inhibitors specific for different families of proteases (Figure 3). This screen shows that the serine protease inhibitors PMSF and leupeptin inhibit the activity to different extends (Figure 3A, lanes 5 and 9). Given that some cysteine proteases can also be inhibited by these compounds, we tested more cysteine protease inhibitors but none of them blocked the activity (Figure 3B). Furthermore, Figure 3A shows that pefacbloc SC plus, which reacts only with the nucleophilic serine in the catalytic site of proteases, can robustly inhibit the H3 endopeptidase activity (lane 12). Together, these results strongly suggest that the activity purified from yeast extracts is a serine protease.

Bottom Line:
Induction of gene expression in yeast and human cells involves changes in the histone modifications associated with promoters.A truncated H3 product is not generated in yeast carrying a mutation of the endopeptidase recognition site (H3 Q19A L20A) and gene induction is defective in these cells.These findings identify clipping of H3 tails as a previously uncharacterized modification of promoter-bound nucleosomes, which may result in the localized clearing of repressive signals during the induction of gene expression.

ABSTRACTInduction of gene expression in yeast and human cells involves changes in the histone modifications associated with promoters. Here we identify a histone H3 endopeptidase activity in Saccharomyces cerevisiae that may regulate these events. The endopeptidase cleaves H3 after Ala21, generating a histone that lacks the first 21 residues and shows a preference for H3 tails carrying repressive modifications. In vivo, the H3 N terminus is clipped, specifically within the promoters of genes following the induction of transcription. H3 clipping precedes the process of histone eviction seen when genes become fully active. A truncated H3 product is not generated in yeast carrying a mutation of the endopeptidase recognition site (H3 Q19A L20A) and gene induction is defective in these cells. These findings identify clipping of H3 tails as a previously uncharacterized modification of promoter-bound nucleosomes, which may result in the localized clearing of repressive signals during the induction of gene expression.